Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually and is highly associated with animal contamination due to the natural reservoir of ruminant gastrointestinal tracts. Free STEC-specific bacteriophages against STEC strains are also commonly isolated from fecal-contaminated environment. Previous studies have evaluated the correlation between the prevalence of STEC-specific bacteriophages and STEC strains to improve animal-associated environment. However, the similar information regarding free STEC-specific bacteriophages prevalence in produce growing area is lacking. Thus, the objectives of this research were to determine the prevalence of STEC-specific phages, analyze potential effects of environmental factors on the prevalence of the phages, and study correlations between STEC-specific bacteriophages and the bacterial hosts in pre-harvest produce environment. Surface water from 20 samples sites was subjected to free bacteriophage isolation using host strains of both generic E. coli and STEC (O157, six non-O157 and one O179 strains) cocktails, and isolation of O157 and non-O157 STEC strains by use of culture methods combined with PCR-based confirmation. The weather data were obtained from weather station website. Free O145- and O179-specific bacteriophages were the two most frequently isolated bacteriophages among all (O45, O145, O157 and O179) in this study. The results showed June and July had relatively high prevalence of overall STEC-specific bacteriophages with minimum isolation of STEC strains. In addition, the bacteriophages were likely isolated in the area—around or within city—with predominant human impact, whereas the STEC bacterial isolates were commonly found in agriculture impact environment. Furthermore, there was a trend that the sample sites with positive of free STEC bacteriophage did not have the specific STEC bacterial hosts. The findings of the study enable us to understand the ecology between free STEC-specific phages and STEC bacteria for further pre-harvest food safety management in produce environment.

Investigation of prevalence of free Shiga toxin-producing Escherichia coli (STEC)-specific bacteriophages and its correlation with STEC bacterial hosts in a produce-growing area in Salinas, California

January
Investigation of prevalence of free Shiga toxin- producing Escherichia coli (STEC)-specific bacteriophages and its correlation with STEC bacterial hosts in a produce-growing area in Salinas, California
Yen-Te Liao 0 1
Irwin A. Quintela 0 1
Kimberly Nguyen 0 1
Alexandra Salvador 0 1
Michael B. Cooley 0 1
Vivian C. H. Wu 0 1
0 Produce Safety and Microbiology Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, California, United States of America, 2 School of Food and Agriculture, University of Maine , Orono, Maine , United States of America
1 Editor: A. Mark Ibekwe, USDA-ARS Salinity Laboratory , UNITED STATES
Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually and is highly associated with animal contamination due to the natural reservoir of ruminant gastrointestinal tracts. Free STEC-specific bacteriophages against STEC strains are also commonly isolated from fecal-contaminated environment. Previous studies have evaluated the correlation between the prevalence of STEC-specific bacteriophages and STEC strains to improve animal-associated environment. However, the similar information regarding free STEC-specific bacteriophages prevalence in produce growing area is lacking. Thus, the objectives of this research were to determine the prevalence of STEC-specific phages, analyze potential effects of environmental factors on the prevalence of the phages, and study correlations between STEC-specific bacteriophages and the bacterial hosts in pre-harvest produce environment. Surface water from 20 samples sites was subjected to free bacteriophage isolation using host strains of both generic E. coli and STEC (O157, six non-O157 and one O179 strains) cocktails, and isolation of O157 and non-O157 STEC strains by use of culture methods combined with PCR-based confirmation. The weather data were obtained from weather station website. Free O145- and O179-specific bacteriophages were the two most frequently isolated bacteriophages among all (O45, O145, O157 and O179) in this study. The results showed June and July had relatively high prevalence of overall STEC-specific bacteriophages with minimum isolation of STEC strains. In addition, the bacteriophages were likely isolated in the areaÐaround or within cityÐwith predominant human impact, whereas the STEC bacterial isolates were commonly found in agriculture impact environment. Furthermore, there was a trend that the sample sites with positive of free STEC bacteriophage did not have the specific STEC bacterial hosts. The findings of the study enable us to understand the ecology between free STEC-specific phages and STEC bacteria for further pre-harvest food safety management in produce environment.
-
Data Availability Statement: The raw data used
for both Fig 2 and Fig 3 are within the Supporting
Information files. In addition, our findings indicate
the isolation of STEC strains, and some sample
sites may be in close proximity to farms. Since the
data were only used for research purpose and the
isolation of STEC strains were likely derived from
wild animals, we have requested not to reveal the
geographical information. Data queries can be
Funding: This work was supported by USDA-ARS
CRIS projects 2030-42000-050-00D.
Introduction
Shiga toxin-producing E. coli (STEC) strains are foodborne pathogens that can cause serious
human illness, such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura.
High mortality is usually found in the population of the elders and children under 5 years of age.
Because ruminants' gastrointestinal tracts are the reservoir for the pathogens, the diseases pri
marily developed through consumption of undercooked and tenderized meat, particular beef
products [
1
]. Nevertheless, during fall 2006, a foodborne outbreak associated with consumption
of contaminated spinach occurred in several states. More than 200 victims were diagnosed with
infection of E. coli O157:H7, and 3 people died [
2
]. In 2011, a catastrophic outbreak occurred in
Germany associated with STEC contamination on fenugreek sprouts, and more than 3,000 cases
of human infection were recorded with 850 patients developing HUS and 53 died [
3
]. Ever since
the concerns regarding STEC infection has been raised on the produce safety. Produce
contamination is a particularly serious situation since it is generally consumed raw.
Although produce can be easily contaminated at any point where pathogens are present in
the production line, surface water, such as rivers or lakes, as a starting source of contamination
in pre-harvest environment, is responsible for the spread of the various foodborne pathogens,
including STEC, to produce through irrigation. Surface water also leads to infection of wildlife,
which may lead to produce contamination due to deposition of feces in the field [
4
]. Wildlife,
runoff from domestic animal facilities, and sewage are the major sources contributing to
contamination of surface water [5]. In the meanwhile, bacteriophages (or phages) that are lytic
against enteric bacteria, such as STEC, were also found to be key elements of intestinal
microbiota of humans and other animals [
6, 7
]. Previous studies isolated free bacteriophages against
E. coli (also known as coliphages) or against STEC strains (or STEC-specific phages) from the
environments highly contaminated with feces of human or animal origins. For example,
previous research focused on isolation of free STEC-specific phages, including serogroups of O26,
O111, and O157, from cattle manure [
8
]. Their findings indicated that these phages were free of
stx, hlyA, and eaeA genes and were good candidates as bio-control agents to prevent STEC
contamination. The research conducted by Muniesa and Jofre with regard to prevalence of the
bacteriophages against E. coli O157:H7 in sewage contaminated with fecal matters showed that the
free phages infecting E. coli O157:H7 strains also harbored stx2 genes and were commonly
found in the sewage collected from different European countries [
9
]. Imamovic et al. evaluated
different sources of water with regard to the prevalence of coliphages encoding stx2 genes (or
Stx2 phages) and found that the phages were positive in 70% and 90% of urban sewage and
animal wastewater samples, respectively [
10
]. In addition, the authors indicated that these free Stx
phages might pose a risk for the spread of stx genes among bacterial populations, and could
result in emergence of new pathogens. These research findings indicate that there were various
coliphages or STEC-specific phages in the animal-associated environment, and each type of
phage might have different roles with regard to the bacterial hosts and the environment.
However, similar information is lacking with regard to the produce production environment.
A study was conducted by U.S. Department of Agriculture research team to evaluate the
prevalence of STEC strains and other foodborne pathogens and the persistence of pathogen
subtypes in a produce central area [
11
]. The findings demonstrated that foodborne pathogens,
including STEC were introduced into produce pre-harvest environment from wildlife and
animal operations and were likely disseminated through surface water. Recently, the same
research team also conducted another study using male-specific coliphages, one group of E.
coli infecting phages, to track the source of fecal contamination from surface water in
proximity to produce-intense regions [
12
]. Their findings could not correlate fecal pollution to the
prevalence of male-specific phages, nor did it provide any information regarding whether or
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not the phages harbored stx genes, and its host range. In addition, the results showed that both
non-O157 STEC and O157 STEC strains were isolated in the environment; however, the
prevalence of free phages against O157 and non-O157 STEC was not determined [
12
]. So far, no
published study aiming at the prevalence of free STEC-specific phages in produce-growing
area is found. There is also a need to evaluate the potential environmental factors that may
affect the prevalence free STEC-specific phages and to determine its association with STEC
strains in pre-harvest produce area in order to improve safety of produce-growing area.
Therefore, the objectives of this research were to 1) determine the prevalence of STEC-specific
phages, to 2) analyze potential effects of environmental factors on the prevalence of the phages,
and to 3) study correlations between STEC-specific bacteriophages and the bacterial hosts in
pre-harvest produce environment.
Materials and methods
Sampling area and sample collection
The sample sites within a heavy crop-growing area in Salinas Valley, CA were selected based on
accessibility and previous experience. A total of 131 samples from 20 watershed sites were
processed and categorized into several groups based on the potential for impact by human or
agriculture activities. The sample sites labeled black acronym on the map indicated predominant
agriculture impact and those with red acronym labeling indicated human impact in Fig 1. Since
produce was grown throughout the Salinas valley, all 20 sample sites are impacted by agriculture,
to a degree. However, sites SABSAL, SLUSAL and CHUCRR were located furthest up stream, in
or near the Salinas River and sites QUAOSR, ZABOSR, TOWOSR, GABOSR, GABCRA, and
GABHER were up stream, along the eastern side of the valley and in several tributaries feeding
Carr Lake, within the city of Salinas. Importantly, cow calf operations were frequently observed
on the hills above these sites. Thus, these sites were classified as agriculture-impacted sites. In
contrast, the sites NATCAR, ALICAR, CAROUT, and RECVIC were located in Salinas City and
were classified as human impact because of direct impact by human activities. Although the sites
ESPESP, TEMPRE, TEMMOL, and OLSMON were along the Tembladero and Espinosa Sloughs
near Castroville, where crop farms were located, the areas were also classified as human-impacted
due to their location, downstream from Salinas and Castroville. Likewise, sites SALDAV,
SAL
BLA, and SALMON, downstream on the Salinas River, were also classified as human impact due
to runoff from several smaller townships along El Toro Creek and the Salinas River.
The samples collected from May to September 2016 were used for the isolation of both free
STEC-specific phages and STEC strains. Water samples were collected using Moore swabs
submerged in surface water for approximately 24h to collect sediment and microorganisms
flowing through the swabs. The swab samples were placed in sterile sampling bags and stored
on ice during transport to the ARS laboratory at Albany, CA. Due to lack of water, not every
site was sampled during the sampling interval. Upon arrival, the swabs were added to 500 ml
sterile water, followed by homogenization by hand for 20s. An aliquot of water (20mL) eluted
from the swabs was stored at 4ÊC prior to further phage analysis. The majority of the sample
(300mL), including the swab and debris, was used for STEC bacterial isolation. The remainder
was used to isolate other pathogens in research not described here.
STEC strain isolation
The STEC strains isolation protocol used in this study was described previously [13]. In brief,
the swab including rinsate was enriched with TSB at 25ÊC for 2h and 42ÊC for 8h. For O157
STEC, the enrichment was subjected to immunomagnetic separation (IMS), followed by plat
ing on selective media, Sorbital MacConkey (Difco Labs: Detroit, MI) with cefixine (0.05μg/
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Fig 1. Geographical location of the watershed sites where the samples were collected in the area of
Salinas Valley. Sample sites are labeled with a six-letter acronym in black type (locations with agriculture
impact) or red type (locations with human impact). Insert is an expanded view of the area near the city of
Salinas.
mL; Introgen/Dynal) and tellurite (2.5μg/mL; Introgen/Dynal) (CT-SMAC) and Rainbow
Agar O157 (Biolog, Hayward, CA) containing novobiocin (20μg/mL; Sigma-Aldrich) and
tellurite (0.8μg/mL; Introgen/Dynal) (NT-RA), for isolation. Subsequently, the presumptive
colonies were screened for the presence of rfbE gene by polymerase chain reaction (PCR) assay. As
for non-O157 STEC, similar methods were used, including IMS, for isolation. A parallel
isolation method included PCR screening for stx genes and was conducted immediately after
sample enrichment. Subsequently, the stx-positive enrichments were plated on CHROMagar O157
(DRG International, Mountainside, New Jersey). The presumptive colonies were then
confirmed for the presence of stx genes by PCR. The serotypes of the isolated STEC strains were
determined by enzyme-linked immunosorbent assay (ELISA), and confirmed by E. coli
Reference Center at Penn State University.
Bacterial host strains for phage isolation
Four E. coli strains lacking stx1 and stx2 genes, including ATCC13706, ATCC43888, DH5, and
MG1655, obtained from U.S. Department of Agriculture (USDA) Agricultural Research Service (ARS) Western Regional Research Center (WRRC), were used for isolating bacteriophage
4 / 12
Source
water
water
water
water
water
cattle feces
water
cattle feces
cattle feces
trough water
cattle feces
water
cattle feces
water
water
water
water
water
water
cattle feces
cattle
cattle
cattle
cattle
water
water
water
water
trough water
harboring stx genes. Four strains per serogroup of O26, O45, O103, O111, O121, O145 and
O157, as well as one O179 strain previously isolated by USDA ARS WRRC, were also used for
phage isolation, as well as host range test (Table 1). Some STEC strains previously isolated from
water samples in Salinas Area were used to increase free phage isolation, and some isolated from
different environmental samples, such as cattle feces and trough water, were also used as hosts
to increase the variety of strains in this study. The overnight cultures of the selected host strains
Ðnon-pathogenic E. coli and STECÐwere prepared by inoculating each sterile tube containing
5ml of tryptic soy broth (TSB, Difco, Becton Dickinson, Sparks, MD) with a 1μl-loop of the indi
vidual strain culture and incubating overnight at 37ÊC. The strain cocktail for either
non-pathogenic or STEC strains was prepared by mixing 0.1ml of each overnight culture immediately
prior to use. Both cocktails of non-pathogenic E. coli and STEC were used separately to inoculate
the water samples for the isolation of free STEC-specific phages.
Phage isolation and purification
Water samples were centrifuged at 4,000xg for 15min to get rid of sediments. An aliquot of 10ml water sample was added into two sterile tubes with 30ml TSB supplemented with 5mM
5 / 12
CaCl2, and subsequently inoculated with 2.8ml and 0.4ml of respective STEC and non-patho
genic E. coli cocktails, respectively. The sample-culture mixers were left at room temperature
for 15min prior to incubation in a bench top incubator with 150rpm shaking (Sheldon
Manufacturing, Inc., Cornelius, OR) at 37ÊC for 48h. After incubation, chloroform at 4% v/v
concentration was added into each enrichment tube and homogenized by use of a
multi-purpose tube rotator (Thermo Fisher Scientific, Waltham, MA) at room temperature for 30min to
kill both bacterial hosts and background flora. The supernatants were obtained after
centrifugation at 8,000xg for 10min.
For confirming the presence of free STEC-specific phages and its host specificity, spot assay
was conducted with the supernatants obtained from the enrichments on the selected host
strains (STEC and non-pathogenic E. coli). Briefly, an aliquot of 0.2ml bacterial overnight
culture was mixed with 10ml molten tryptic soy agar (TSA, Difco, Becton Dickinson, Sparks,
MD) amended with 5mM Calcium Chloride (CaCl2) to enhance phage adsorption onto
bacterial surface and poured into a sterile Petri plate. Subsequently, ten microliters of each
supernatant were spotted on the strain-inoculated TSA plate and incubated at 37ÊC for 24h. As a
result, the positive samples showed a circular clearing zone, indicating the presence of phages
lytic against the tested strain. The supernatants, which were used for spot assay and had
positive results, were further enriched with each of the spot test-positive strains by adding 0.1ml of
supernatant with 0.5ml of the overnight bacterial culture supplemented with CaCl2 at 5mM in
5ml of TSB and incubated at 37ÊC for 24h. After incubation, the propagated phage solutions
(also known as lysates) were centrifuged at 8,000xg for 10min to get rid of bacterial debris.
Subsequently, the phage lysates were subjected to single layer plaque assay for phage
separation. Briefly, the phage lysate was serial diluted, followed by mixing 0.2ml of the diluted lysate
and 0.3ml overnight bacterial culture in 10ml molten TSA supplemented with CaCl2 prior to
pouring to a sterile Petri dish plate. After incubation at 37ÊC for 24h, three segregated plaques
with different sizes were selected for further purification. Each plaque was enriched with 0.2ml
of the bacterial host culture in 8ml of TSB at 37ÊC for 24h. After enrichment, the phage lysate
was subjected to centrifugation at 8,000xg for 10min and then single plaque assay. Later, one
plaque was selected for two more run of enrichment and plaque assay as describe above for
phage purification. At the end, the phage lysates were filtered through 0.22μm syringe filter
membrane and quantified by use of single layer plaque assay prior to further analysis.
Transmission electron microscopy
Phage lysates were subjected to polyethylene glycol concentration using PEG kit (PEG, BioVi
sion Inc., Milpitas, CA) according to manufacturer's instruction, and morphology of the phage
was examined by transmission electron microscopy (TEM). Briefly, an aliquot of 6μl of phage
sample was applied on copper mesh PLECO grids (Ted Pella Inc., Redding, CA) and incubated
for 1min at room temperature. The copper mesh grid containing phage sample was carefully
blotted on Whatmann filter paper and subjected to negative staining by adding 8μl of 0.75%
Uranyl acetate (Sigma-Aldrich, Darmstadt, Germany) for 30s incubation at room temperature.
The specimen was then examined in a transmission electron microscope (FEI Tecnai G2).
DNA extraction and PCR screening
After phage concentration by PEG, the phage lysates were subject to DNA extraction with
commercial phage DNA extraction kit according to manufacturer's instruction (Norgen
Biotek Corp., Ontario, Canada). Detection of stx genes were investigated by use of PCR with
10μM each of stx1 forward/ reverse primer pairs (5’-CATCGCGAGTTGCCAGAATG-3’/
5’-AATTGCCCCCAGAGTGGATG-3’) and stx2 forward/ reverse primer pairs (5’- GTAT
6 / 12
ACGATGACGCCGGGAG-3’/ 5’- TTCTCCCCACTCTGACACCA-3’) [
14
]. PCR master mix
(Promega Corp., Madison. WI) was added to the sample with a total reaction volume of 50μl,
and thermal parameters for denaturing, annealing, and extension temperatures of stx1 and
stx2 genes are 95ÊC, 56ÊC, 72ÊC and 95ÊC, 58.1ÊC, 72ÊC, respectively, for 28 cycles. The target
amplicons were 119bp for stx1 and 104bp for stx2. The PCR products were subjected to
electrophoresis using 1.5% agarose gel with a 100bp DNA ladder at 100 voltages for approximately
2h and stained with 3x GelRed prior to visualization on the Alpha Imager UV gel box.
Collection of environmental data
The information of rain precipitation and solar radiation were collected from the website of
California Irrigation Management Information System (CIMIS). The rain precipitation data
for each sample site were collected during 5 days ahead through the day of samples collection,
and the solar radiation data was collected only on the day of sampling for statistical analysis.
These environmental factors, rain precipitation and solar radiation, were included in this study to evaluate their effects on the prevalence of free STEC-specific phage because these were two of the major factors that attributed to the growth of produce.
Data analysis
Statistical analysis was performed using JMP1 (Version 12.0.1, SAS Institute Inc., Cary, NC).
One-way analysis of variance (ANOVA) tests were used to evaluate correlation between envi
ronmental factors (rain precipitation or solar radiation) and STEC-specific phage isolation.
Student T test was used to evaluate different environmental impacts (human vs. agriculture) on the phage isolation.
Results and discussion
This is the first study focusing on the prevalence of free STEC-specific bacteriophages at a pre
harvest produce area in Salinas, CA. In order to increase isolation of the free STEC-specific
phages, both non-pathogenic and pathogenic STEC cocktails were used. Furthermore, the use
of generic E. coli would also facilitate isolation of the phages harboring stx genes in this study.
A previous study utilized non-pathogenic O157 strain to isolate Stx-phages from waste water
and river water, and the findings showed that stx2 was more prevalent than stx1 in the phages
isolated from the aquatic environment [
15
]. In addition, the authors revealed that the isolated
Stx-phage were at low levels in their study. On the contrary, in current study, none of the
STEC-specific bacteriophages were positive of either stx1 or stx2 gene. Therefore, there was
less likely that the phages isolated in this study could serve as potential mobile genetic elements
for transferring virulence genes in the environment [
16
]. The current results with no detection
of stx genes in the phages could be due to low levels of fecal contamination around
producegrowing environment in Salinas, which was correlated with the findings of previous research,
in that the authors failed to track fecal contamination in Salinas area using male-specific
coliphages as indicators due to scarcity of STEC strain isolation [
12
].
In this study, a number of STEC-specific phages and STEC strains isolated from various
sample sites in Salinas area are indicated in Fig 2 (131 samples from 20 watershed sites). A
total of 9.9% (n = 13) of the water samples collected in this study were positive of free lytic
bacteriophages against STEC strains, including serogroups of O45, O145, O157 and O179, and
were distributed among 8 different watershed sites. The O145- and O179-specific phages were
most frequently isolated (4 different sites) in this study, followed by O157-specific phages (2
sites), and O45-specific phages (1 site). Interestingly, both O145- and O179-specific phages
were likely found in the area close to city where human impact was predominant; i.e., the site
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Fig 2. Summary of phage and STEC isolation data from water samples collected from Salinas, CA. The acronym is used
for each sample site with the number of water samples collected. Red oval shape with red letter indicates isolation of
STECspecific phage, and green rectangular shape with green letters indicates STEC bacterial isolation.
ªCAROUTº was positive for three different STEC-specific phages (O145, O157 and O179).
Although not statistically different, the results showed that the naturally occurring
STEC-specific phages were more likely isolated in the human impact-associated area rather than in
agricultural impact region (P>0.05). Additionally, the sample sites with isolation of O145-specific
phages (GABCRA, CAROUT, SALMON, OLSMON) were either located in the same flow
path (GABCRA & CAROUT) or affected by nearby merging river branch, indicating that the
phages were likely to be disseminated through river flow. Similar phenomenon was also found
on some of the free O179-specific phages positive sites (CAROUT, ESPESP, TEMPRE). On
the other hand, though not subjected to analysis, more sample sites in the agriculture-impact
area were positive of the STEC strains than those in human-impact area that could be due to
cow calf operation or wild animal activities. Furthermore, O157 STEC strains were the most
predominant bacterial isolates found in this study, both in areas with human impact and
agriculture impact, and the river might also attribute to the spread of the pathogens. Although
some sample sites were positive of both STEC strains and STEC-specific phages, it seemed that
the sites with presence of STEC-specific bacteriophages did not have the STEC bacterial hosts
isolated. The findings could be derived from the mitigating effects of these lytic bacteriophages
against the STEC bacterial population because the spot test used at the early stage of the phage
isolation showed that the phages were able to produce lysis zone against some of the top six
non-O157 and O157 STEC strains selected in this study. Hallewell et al. studied the prevalence
of E. coli O157:H7-infecting phages in the cattle that shed the O157 strains. The authors found
8 / 12
Fig 3. Samples with positive of STEC-specific phages and STEC strains throughout sampling period displayed by
sample month.
the cattle that shed O157:H7 the most had lower prevalence of O157-specific phages, whereas
the low O157:H7 shedder cattle had higher prevalence of the phages [
17
]. The antibacterial
effect of the free STEC-specific phages isolated from either produce-growing environment or
animal-associated habitats showed the potential of these free lytic phages as bio-control agents
to prevent STEC contamination.
In current study, the samples used for the isolation of free phages and STEC strains were
collected from May to September 2016, in which covered late spring, summer and early fall
seasons. The results showed that July had the highest overall prevalence of STEC-specific
phages regardless of types of environment (human impact vs. agriculture impact) of the sample
sites without any isolation of STEC bacteria, followed by June with the second highest
prevalence of free phages (Fig 3). However, in May and August, STEC strains were frequently
isolated from the sample sites, while no free phage was found. It was interesting to observe in this
study that the more free STEC-specific phages were present, the less isolation of STEC strains
was obtained during the same period of time. Ravva et al. evaluated the fecal contamination in
Salinas area by use of male-specific coliphages, and their findings indicated that the DNA
coliphages were predominantly isolated in summer time, which was consistent with our results
regarding prevalence of STEC-specific phages [
12
]. In addition, a previous study evaluated the
seasonal prevalence of O157 STEC and non-O157 STEC in Salinas area and found that overall
prevalence of STEC strains was lower during summer and fall periods [
11
]. Their findings
were correlated with our findings, in that higher prevalence of STEC-specific phage might
account for the low prevalence of STEC bacterial isolates during summer time.
The results showed that, though not statically significant (P = 0.079), the sample sites with
higher rain precipitation were likely to have higher prevalence of free phages (Table 2). This
could be due to higher water levels by rain precipitation resulting in the spread of phages
through affluent river flow all over the area, which was consent to the earlier observation that
river flow may play an important part of facilitating phage dissemination. On the other hand,
the current results also indicated that there was no significant difference (P = 0.717) between
solar radiation and phage isolation, with the average radiation intensity of 476 Ly and 465 Ly
at overall phage positive and negative sample sites, respectively (Table 2). Previous study
9 / 12
Environmental factorsa
aLeast-squares means were calculated within each factor, and values within each factor with the same letter (A, B) are not significantly different (P>0.05).
bData presented are mean ± SD (standard deviation).
indicated that the bacteriophage Lambda was resistant to UV radiation in a lab setting and it
was able to remain viable under high-dose UV (300mJ/cm2) [
18
]. Although this study did not
design to evaluate the environmental fitness to the bacteriophages, the current data likely
suggest that the environmental factors, rain precipitation and solar radiation, had minimal effect
on the prevalence of free STEC-specific phages.
Moreover, the electron microscopy showed that these STEC-specific phages had different
morphologies belonging to Siphoviridae or Myoviridae family (Fig 4). Although phage
morphology was not tightly correlated with specific serogroup of the host strains, the results
revealed that both O45- and O157-specific phages that were isolated by use of generic E. coli
Fig 4. The morphology of the STEC-specific phages isolated from Salinas area. (A) O157-specific bacteriophage, (B)
O145-specific bacteriophage, (C) O45-specific bacteriophage, (D) O179-specific bacteriophage.
10 / 12
cocktail were likely to be Myoviridae family, while O145- and O179-specific phages isolated by
STEC cocktail were Siphoviridae family.
In conclusion, this is the first study to determine the prevalence of STEC-specific phages
against serogroups of O26, O45, O103, O111, O121, O145, and O157 in pre-harvest produce
environment in the US. The present study discovered STEC-specific bacteriophages, including
serogroups of O45, O145, O157 and O179, which did not harbor stx genes. The findings
suggest that the free STEC-specific phages were commonly isolated in the locations close to city or
area with predominant human impact, and were likely disseminated from one place to another
through river stream. The season, particular summer, contributed to higher prevalence of free
STEC-specific phages in pre-harvest produce area in this study, even though rain precipitation
and solar radiation did not have significant influence. Most interestingly, these results possibly
suggest the mitigating effect of the free STEC-specific phages on its STEC bacterial hosts in the
produce-growing area, i.e., the prevalence of lytic STEC-specific bacteriophages is negatively
correlated with STEC strains in the pre-harvest produce environment. Further analysis is
needed to evaluate effects of other environmental factors or the interaction of the
environmental factors in long-term sampling plan.
Acknowledgments
We would like to thank Anne Bates and Diana Carychao from PSM for help with the host strain isolation and selection, and Delilah Wood from TEM lab for the assistance with bacteriophage TEM image in this project.
Author Contributions
Conceptualization: Vivian C. H. Wu.
Data curation: Yen-Te Liao, Vivian C. H. Wu.
Funding acquisition: Vivian C. H. Wu.
Investigation: Vivian C. H. Wu.
Michael B. Cooley.
Project administration: Vivian C. H. Wu.
Supervision: Vivian C. H. Wu.
Writing ± original draft: Yen-Te Liao.
Methodology: Yen-Te Liao, Irwin A. Quintela, Kimberly Nguyen, Alexandra Salvador,
Writing ± review & editing: Michael B. Cooley, Vivian C. H. Wu.
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5.
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